Profiling of APEX-NLS and APEX-PMLca associated regions
Ontology highlight
ABSTRACT: To address specificity of ALaP-seq for PMLwt, we performed genome-wide profiling of genomic regions associated with NLS-APEX or PMLca-APEX. Cells were treated with H2O2 to trigger labeling of chromatin with biotin (H2O2+). Experimental negative control for PMLca (H2O2-), where the H2O2 treatment was omitted, was also analyzed.
Project description:Although stress granules (SGs) are composed of a stable core structure, they are surrounded by a dynamic shell with assembly, disassembly, and transitions of proteins and RNA between the core and shell. Different SGs have distinct proteins and RNA constituents, which raises the possibility that different SGs might perform different biological functions. The RNA compositions of DDX3X- or DDX3Y-positive SGs are not known, nor is it known what differential effects DDX3X or DDX3Y may exert on these RNA components. To understand the biological function and the compositional differences between DDX3X- or DDX3Y-positive SGs, we first determined the RNA constituents in these SGs using an adapted Ascorbate-peroxidase (APEX)-based proximity labeling method. APEX converts exogenously supplied biotin-phenol (BP) to biotin-phenoxyl radicals, which in turn covalently labels protein and RNA in nanometers. APEX-mediated biotinylation has been widely used in studies of protein-protein interactions and recently in studying protein-RNA interactions. Here, we applied the APEX-mediated biotinylation in DDX3X and DDX3Y SGs to dissect their different RNA composition.
Project description:Proximity labeling approach to identify protein inside nuclear envelope blebs arising in Torsin-deficient HeLa cells. WT and TorsinKO cells were engineered to express MLF2-APEX2 fusion. APEX reaction was carried out via 1 mM H2O2 for 1 minute in the presence of biotin phenol. Biotinylated protein were captured via streptavidin beads. Each APEX reaction was accompanied by an untreated (no H2O2) control.
Project description:We present MERR APEX-seq, a method for newly transcribed RNAs subcellular profiling combined metabolic incorporation of electron-rich ribonucleosides, 6-thioguanosine and 4-thiouridine, with the peroxidase-mediated RNA labeling method, APEX-seq. MERR APEX-seq offers both high spatial specificity and high coverage in the mitochondrial matrix and at the endoplasmic reticulum membrane. Application of MERR APEX-seq at nuclear lamina of human cells reveals that the mRNA components tend to encode for transcripts processing related proteins. MERR APEX-seq with high spatial specificity and high coverage could be widely used to expand our knowledge of RNA localization and function at subcellular compartments.
Project description:We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
Project description:Stress granules are dynamic non-membrane bound organelles made up of untranslating messenger ribonucleoproteins (mRNPs) that form when cells integrate stressful environmental cues resulting in stalled translation initiation complexes. Although stress granules dramatically alter mRNA and protein localization, understanding these complexes has proven to be challenging through conventional imaging, purification, and crosslinking approaches. We therefore developed an RNA proximity labeling technique, APEX-Seq, which uses the ascorbate peroxidase APEX2 to probe the spatial organization of the transcriptome. We show that APEX-Seq can resolve the localization of RNAs within the cell and determine their enrichment or depletion near key RNA-binding proteins. Matching both the spatial transcriptome using APEX-seq, and the spatial proteome using APEX-mass spectrometry (APEX-MS) provide new insights into the organization of translation initiation complexes on active mRNAs, as well as revealing unanticipated complexity in stress granule contents, and provides a powerful approach to explore the spatial environment of macromolecules.
Project description:Several studies have shown that plant hormones play key roles during legume-rhizobia symbiosis. For instance, auxins can induce formation of nodule-like structures (NLS) on legume roots in the absence of rhizobia. Furthermore, these NLS can be colonized by nitrogen-fixing bacteria, which favor nitrogen fixation compared to regular roots and subsequently increase plant yield. Interestingly, auxin also induces similar NLS in cereal roots. While several genetic studies have identified plant genes controlling NLS formation in legumes, no studies have investigated the genes involved in NLS formation in cereals. In this study, we performed a comprehensive RNA sequencing experiment to identify genes differentially expressed during NLS formation in rice at different stages and identified several promising genes for control of NLS based on their biological and molecular functions.
Project description:In this study we treated Brachypodium distachyon roots with synthetic auxin, 2,4-D, to induce nodule-like structures (NLS) and performed RNA-seq to assess transcriptome changes during NLS formation.
Project description:An integrating APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput ma